CN109797219A - Detect application and the kit of the reagent of ABRACL expression - Google Patents
Detect application and the kit of the reagent of ABRACL expression Download PDFInfo
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- CN109797219A CN109797219A CN201910016197.0A CN201910016197A CN109797219A CN 109797219 A CN109797219 A CN 109797219A CN 201910016197 A CN201910016197 A CN 201910016197A CN 109797219 A CN109797219 A CN 109797219A
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Abstract
The invention belongs to field of biotechnology, it is related to detecting application and the kit of the reagent of ABRACL expression.Application of the reagent in preparation of the preparation for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis more particularly, to detection ABRACL expression and a kind of for Computer-aided Diagnosis of Breast Cancer and/or the kit of patient with breast cancer's Index for diagnosis.Clinical sample testing result shows that ABRACL expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer；And ABRACL high expression is unfavorable for patient with breast cancer's overall survival (P=0.035).The reagent for detecting the changes in gene expression can be used for Prognosis in Breast Cancer or diagnosis, treatment.
Description
Technical field
The invention belongs to field of biotechnology, use more particularly, to the reagent of detection ABRACL expression in preparation
Application and a kind of breast cancer auxiliary that is used in the preparation of Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis are examined
Disconnected and/or patient with breast cancer's Index for diagnosis kit.
Background technique
Breast cancer is most commonly seen gynecologic malignant tumor.Global cancer epidemiology statistical data shows, the world in 2012
In range breast cancer new cases sum 1677000, be only second to lung cancer the second high-incidence tumour and women disease incidence and
The highest malignant tumour of the death rate.China's breast cancer incidence is up to 25.89/10 ten thousand, accounts for about all female malignant morbidities
The 16.83% of rate.Moreover, China's breast cancer illness number and new diagnosis patient's number are just being increasing year by year in global ratio, and
Show morbidity rejuvenation trend, the physical and mental health for seriously affecting women, even threat to life.Therefore, pathogenesis of breast carcinoma mechanism
Research with prognosis is of far-reaching significance to patient with breast cancer.Breast cancer is in origin of cell, Histological Study, disease classification, clinical table
Existing, therapeutic response and metastatic potential etc. all show great complexity and heterogeneity, limit Prognosis in Breast Cancer mark
The popularity of will object application.Therefore, there is an urgent need to develop more targeted prognostic marker for breast cancer, to meet clinical need
It asks.
With quickling increase for the high-throughput data of multiple groups, some molecular biology markers are found and mammary gland carcinogenesis
Related with prognosis, this makes it possible more acurrate, effectively diagnosis and treatment breast cancer.
Summary of the invention
The object of the present invention is to provide a kind of new prognostic marker for breast cancer ABRACL, thus further provide for detecting
The reagent of ABRACL expression is in preparation in the preparation of Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis
Using and it is a kind of for Computer-aided Diagnosis of Breast Cancer and/or the kit of patient with breast cancer's Index for diagnosis.
ABRACL also known as C6orf115, is positioned at 6q24.1, includes 4 exons, and Gene ID:58527 encodes egg
White matter is positioned at cytoplasm and nucleus.Currently, the effect in relation to ABRACL gene in breast cancer occurrence and development has not been reported.
The present inventor passes through cancer and the high-throughput data mining of oncogene map (Cancer Genome Atlas, TCGA)
It was found that ABRACL high expression is unfavorable for patient with breast cancer's prognosis, and by collecting clinical breast cancer clinical samples and follow-up information,
Further Prognostic of the verifying ABRACL differential expression to patient with breast cancer.
To achieve the goals above, the reagent that the first aspect of the present invention provides detection ABRACL expression is used in preparation
Application in the preparation of Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis.
Further, the detection ABRACL expression includes gene expression dose and/or the detection for detecting ABRACL
The protein expression level of ABRACL.
More specifically, the method for the detection ABRACL expression includes: by RT-qPCR method detection breast cancer and cancer
The expression quantity of ABRACL in tissue；ABRACL mrna expression amount in breast cancer and cancer beside organism is detected by molecular probe technology；
ABRACL expressing quantity variation in breast cancer tissue is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent of the detection ABRACL expression is the oligonucleotide for targeting ABRACL DNA sequences encoding
Probe, PCR primer, or to target the antibody of ABRACL.
In accordance with the present invention it is preferred that the reagent of the detection ABRACL expression is with SEQ ID NO:1 and SEQ
The real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in ID NO:2.
5 '-GCTGATGGAAAGTTAAGCGTGA-3 ', SEQ ID NO:1；
5 '-TGCTTCAAAGAGGTTGGCAC-3 ', SEQ ID NO:2.
The second aspect of the present invention provides a kind of examination for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis
Agent box, the kit include: the reagent for detecting ABRACL expression.
Further, the reagent of the detection ABRACL expression is the oligonucleotide for targeting ABRACL DNA sequences encoding
Probe, PCR primer, or to target the antibody of ABRACL.
Specifically, the reagent of the detection ABRACL expression is with shown in SEQ ID NO:1 and SEQ ID NO:2
The real-time fluorescence quantitative PCR specific primer of nucleotide sequence.
According to the present invention, the kit can also be containing other conventional reagents for real time fluorescent quantitative, preferably
Ground, the kit further includes at least one of following components: Trizol, isopropanol, chloroform, dehydrated alcohol, without RNA enzyme
Water, random primer, 5 × M-MLV buffer, dNTPs, RNase inhibitor, M-MLV reverse transcriptase, have SEQ ID NO:3 and
The ACTB real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in SEQ ID NO:4.
5 '-GGCACCCAGCACAATGAAGA-3 ', SEQ ID NO:3；
5 '-ACTCCTGCTTGCTGATCCAC-3 ', SEQ ID NO:4.
Clinical sample testing result shows that ABRACL expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer；And
ABRACL high expression is unfavorable for patient with breast cancer's overall survival (P=0.035).The reagent for detecting the changes in gene expression can be used for
Prognosis in Breast Cancer or diagnosis, treatment.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its
Its purpose, feature and advantage will be apparent.
Fig. 1 shows the expression of ABRACL in TCGA high throughput data analysis breast cancer.ABRACL table in breast cancer tissue
Up to being significantly higher than normal tissue (P < 0.001).
Fig. 2A -2B shows TCGA high throughput data analysis ABRACL high and expresses the influence survived to patient with breast cancer.
ABRACL high expression is unfavorable for patient with breast cancer's overall survival (P=0.006) (Fig. 2A)；ABRACL high expression be unfavorable for patient without
Diease occurrence deposits (P < 0.001) (Fig. 2 B).
Fig. 3 shows the expression of ABRACL in breast cancer tissue.ABRACL expression is significantly higher than normal in breast cancer tissue
It organizes (P < 0.001).
Fig. 4 shows ABRACL high in breast cancer tissue and expresses the influence survived to patient with breast cancer.ABRACL high expression
It is unfavorable for patient with breast cancer's overall survival (P=0.035).
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe preferred implementations of the invention
Mode, however, it is to be appreciated that may be realized in various forms the present invention without that should be limited by the embodiments set forth herein.It is real
The person that is not specified actual conditions in example is applied, is all carried out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not
Production firm person is indicated, is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
The present embodiment is used to illustrate the expression variation of ABRACL in TCGA high throughput data analysis breast cancer.
1.TCGA high throughput data analysis process:
Login TCGA portal website UALCAN (http://ualcan.path.uab.edu/index.html) homepage, point
It hits " Analysis ", inputs Gene Name " ABRACL ", select TCGA dataset " Breast invasive
Carcinoma ", retrieval are clicked " Expression ", and result is recorded.It is mapped using 6.0 software of GraphPad, statistical method is
T is examined, and P < 0.05 is that difference is statistically significant.
2. result: the expression of ABRACL significantly increases (P < 0.001) compared with normal tissue in breast cancer tissue, as shown in Figure 1.
Embodiment 2
The present embodiment is for illustrating that TCGA high throughput data analyze ABRACL high expression and Prognosis in Breast Cancer relationship.
1.TCGA high throughput data analysis process:
It logs in TCGA portal website cBioPortal (http://www.cbioportal.org/), selects tumour data set
" Breast Invasive Carcinoma (TCGA Provisional) (1093) ", group learn data and select " mRNA
Expression z-Scores (RNA Seq V2RSEM) ", gene input " ABRACL:EXP >=2 ", it retrieves, in popup web page
It clicks " Survival ", records result.Kaplan-Meier method draws survivorship curve, and log-rank examines survivorship curve difference, P
< 0.05 is that difference is statistically significant.
2. result: ABRACL high expression is unfavorable for patient with breast cancer's overall survival (P=0.006), as shown in Figure 2 A；
ABRACL high expression is unfavorable for patient's disease-free survival (P < 0.001), as shown in Figure 2 B.
Embodiment 3
The present embodiment is used to illustrate that the reagent of preparation detection ABRACL expression quantity to be used to prepare the examination of patient with breast cancer's prognosis
Agent box (50 secondary response).
1.Trizol 50.0ml；
2. isopropanol 50.0ml；
3. chloroform 50.0ml；
4. dehydrated alcohol 50.0ml；
5. without RNA enzyme water 5.0ml
6.1.0 50.0 μ l of μM random primer (Random primers)；
7.5 × M-MLV buffer 2.0ml；
8.10.0mM 100.0 μ l of triphosphoric acid base deoxynucleotide (dNTPs)；
50.0 μ l of 9.40U/ μ l RNase inhibitor；
50.0 μ l of 10.200U/ μ l M-MLV reverse transcriptase；
11.ABI 2×PCR Mix 2.0ml；
12.10.0 μM 30.0 μ l of ABRACL real-time fluorescence quantitative PCR specific primer, primer sequence are shown in Table 1；
13.10.0 μM 30.0 μ l of ACTB real-time fluorescence quantitative PCR specific primer, primer sequence are shown in Table 1；
1 fluorescence quantitative RT-PCR primer sequence of table
Embodiment 4
The present embodiment is used to illustrate the detection of clinical breast cancer tissue samples ABRACL.
1. present study carries out under patient's informed consent.57 patient with breast cancer's clinical information are remembered from patient assessment
Record.Breast cancer sample is divided into two parts: a part is freezed immediately in liquid nitrogen, is stored in -80 DEG C until carrying out RNA extraction, separately
It is a part of then be used for histopathological evaluation.
2. Total RNAs extraction in tissue: this experiment carries out in ice bath.30~50mg is taken to organize (fresh or -70 DEG C and liquid nitrogen
The tissue of middle preservation) it sets in 1.5ml centrifuge tube, 1ml Trizol is added and is sufficiently homogenized, is stored at room temperature 5min；Every pipe is added
200 μ l chloroforms acutely mix 30sec, stand 15min, and 4 DEG C of 12000rpm are centrifuged 15min；400 μ l of gentle aspiration supernatant liquid
To in another new centrifuge tube, isometric isopropanol is added, is gently mixed by inversion, 4 DEG C of 12000rpm are centrifuged 10min；Supernatant is abandoned, is added
Enter 75% ethanol wash sediment of 1ml, 4 DEG C of 12000rpm are centrifuged 10min；Supernatant is discarded as far as possible, dries 10min at room temperature,
10 μ l are added without RNA enzyme water in every pipe, dissolve (65 DEG C of dissolution 10-15min).OD260 is measured, RNA concentration is calculated.
RNA (mg/ml)=40 × OD260× extension rate (n)/1000
3. reverse transcription: every 25 μ l reverse transcription system includes 100p mol random primer, 2 μ g total serum IgEs, M-MLV reverse transcriptase 1
0.625 1.25 μ l, 5 × M-MLV buffer of μ l, dNTPs (10mM) of μ l, RNase inhibitor, 5 μ l, no RNA enzyme water polishing to 25 μ
l.Reaction condition are as follows: 37 DEG C of 1h, 95 DEG C of 5min.
4. quantitative PCR: every 20 μ l reaction system includes 2 × PCR Mix, 10 μ l, each 0.4 μ l, cDNA 1 of upstream and downstream primer
μ l, ddH2O 8.2μl.Reaction condition are as follows: 94 DEG C of 2min, 94 DEG C of 15s, 60 DEG C of 40s, 40 circulations.
5.2-ΔΔCTMethod calculates ABRACL relative expression quantity: this experiment detects 57 breast cancer tissues and 18 cancer beside organisms
The relative expression quantity of middle ABRACL changes.ACTB is as reference gene, the target gene ABRACL C that qPCR is measuredTIt is worth and same group
Knit the C of the reference gene ACTB in sourceTValue subtracts each other to obtain Δ CT, then by Δ CTWith control group Δ CTSubtract each other to obtain Δ Δ CT(take cancer
Other sample Δ CTAverage value be Δ CTControl), every group of ABRACL relative expression is calculated using Power function in Excell table
Amount.It is drawn using software GraphPad Prism 6.0, ABRACL differential expression by T check analysis breast cancer and cancer, P < 0.05
There is statistical significance for difference.
6.ABRACL high expression and patient with breast cancer's prognosis: follow-up of patients's time is 1-40 months, successfully receives follow-up trouble
Person's number is 57.It is that height is expressed that ABRACL relative expression quantity, which is higher than 2 times of cancer beside organism's relative expression quantity mean, totally 18,
He is ABRACL low expression, totally 39.Kaplan-Meier method draws survivorship curve, and log-rank examines survivorship curve difference, P
< 0.05 is that difference is statistically significant.
7. result:
Clinical sample testing result shows that ABRACL expression significantly increases (P < 0.001) compared with cancer beside organism in breast cancer, such as
Shown in Fig. 3；And ABRACL high expression is unfavorable for patient with breast cancer's overall survival (P=0.035), as shown in Figure 4.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.
Sequence table
<110>Jiangsu medical profession institute
<120>application and the kit of the reagent of ABRACL expression are detected
<130> BJI1802246JSYY
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gctgatggaa agttaagcgt ga 22
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tgcttcaaag aggttggcac 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggcacccagc acaatgaaga 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
actcctgctt gctgatccac 20
Claims (9)
1. the reagent for detecting ABRACL expression is used for Computer-aided Diagnosis of Breast Cancer and/or patient with breast cancer's Index for diagnosis in preparation
Preparation in application.
2. application according to claim 1, wherein the detection ABRACL expression includes the gene for detecting ABRACL
Expression and/or the protein expression level for detecting ABRACL.
3. application according to claim 1, wherein the method for the detection ABRACL expression includes: the side RT-qPCR
Method detects the expression quantity of ABRACL in breast cancer and cancer beside organism；It is detected in breast cancer and cancer beside organism by molecular probe technology
ABRACL mrna expression amount；ABRACL expressing quantity in breast cancer tissue is detected by immunohistochemistry or Western-Blot
Variation.
4. application according to claim 1, wherein the reagent of the detection ABRACL expression is that targeting ABRACL is compiled
The oligonucleotide probe of code DNA sequence dna, PCR primer, or to target the antibody of ABRACL.
5. application according to claim 4, wherein the reagent of the detection ABRACL expression is with SEQ ID
The real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in NO:1 and SEQ ID NO:2.
6. a kind of for Computer-aided Diagnosis of Breast Cancer and/or the kit of patient with breast cancer's Index for diagnosis, which includes: detection
The reagent of ABRACL expression.
7. kit according to claim 6, wherein the reagent of the detection ABRACL expression is targeting ABRACL
The oligonucleotide probe of DNA sequences encoding, PCR primer, or to target the antibody of ABRACL.
8. kit according to claim 7, wherein the reagent of the detection ABRACL expression is with SEQ ID
The real-time fluorescence quantitative PCR specific primer of nucleotide sequence shown in NO:1 and SEQ ID NO:2.
9. kit according to claim 6, wherein the kit further includes at least one of following components:
Trizol, isopropanol, chloroform, dehydrated alcohol, without RNA enzyme water, random primer, 5 × M-MLV buffer, dNTPs, RNA enzyme inhibit
Agent, M-MLV reverse transcriptase, the ACTB real time fluorescent quantitative with nucleotide sequence shown in SEQ ID NO:3 and SEQ ID NO:4
PCR specific primer.
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